The goal of this project is to determine the mechanism by which cellular signaling pathways target chromatin with a particular focus on signaling mediated through cyclic nucleotides. At gene promoters targeted by various signaling pathways, histones are often found to be modified in response to activation of these pathways. The changes in histone modification influence the activity of these promoters in a variety of ways. In addition, some histone modifications in bulk cellular chromatin are found to be cell cycle dependent. For example, chromatin-associated histone H3 becomes globally phosphorylated just prior to mitosis and is then dephosphorylated upon exit from mitosis. The signaling cascades which lead to both gene-specific and bulk changes in histone modifications are still largely unknown. In the process of studying histone modification at the MMTV promoter in response to signaling pathways, we determined that cAMP signaling causes a drastic reduction in bulk levels of histone H3 phosphorylation in our breast adenocarcinoma cell lines. Further investigation of this phenomenon showed that it is targeted to the mitotic population of phosphorylated histone H3. In fact, cAMP signaling prevents cells from phosphorylating histone H3 at multiple sites due to G2 arrest and inhibition of mitotic entry. Outside of DNA damaging agents, examples of G2 arrest are rare. Unlike normal cells, many cancer cells have been found to be G1 checkpoint deficient, forcing them to rely heavily on the G2 checkpoint to repair genetic damage. This has led some to propose the development of anti-cancer drugs which abrogate the G2 checkpoint. Tumor cells unable to repair genetic damage would eventually undergo apoptosis. Normal cells, which are G1 checkpoint competent, can repair genetic damage prior to DNA replication, and would be far less sensitive to the effects of G2 checkpoint abrogators. Unfortunately candidate targets for such agents are limited because our knowledge of events which occur during the G2 phase of the cell cycle is relatively incomplete. Study of signaling-induced G2 arrest may elucidate novel regulatory mechanisms for G2 progression and identify additional targets for G2 checkpoint abrogation. Our current and future studies will be directed at understanding the molecular events which lead to cAMP-induced G2 arrest and loss of the ability of cells to achieve mitotic levels of H3 phosphorylation. We are particularly interested in determining how cAMP-induced G2 arrest might be different from that induced by DNA damage. Because of its effects on cell cycle progression, we are very interested in the cAMP-induced pathway which leads to this effect. Protein kinase A (PKA) and Epac are thought to mediate most cAMP effects in non-neuronal cells. Interestingly, specific antagonists of PKA do not inhibit cAMP-induced loss of H3 phosphorylation and specific agonists of Epac do not induce it. In collaboration with Dr. Hans-Gottfried Genieser (Biolog Institute, Bremen, Germany) we took a chemistry-based approach to characterize the cAMP receptor which functions in our pathway by screening a large variety of chemically-modified cyclic nucleotides. Through these results we have reached the following conclusions: 1.) the receptor is cAMP-specific and does not bind cGMP, 2.) the cyclic nucleotide binding properties of the receptor are not consistent with PKA, Epac, or the Cyclic Nucleotide-gated Ion Channels (CNGs), and 3.) the activation of the receptor does not correlate with the membrane permeability of various cAMP analogs. In fact, some analogs considered to be membrane impermeable efficiently induced the loss of H3 phosphorylation. This leads us to hypothesize that the receptor may either be extracellular and bind cAMP outside the cell, or intracellular, but coupled to a cyclic nucleotide transporter or pump. Both scenarios imply that cAMP could be inducing the pathwas through extracellular action. Evidence for extracellular action of cyclic nucleotides in mammals has slowly accumulated over the years but a mammalian homolog of the extracellular cAMP receptor in Dictostylium has never been identified. In humans the liver excretes cAMP into the blood and it is thought to act on the kidney from the extracellular space. In addition, there are reports of extracellular effects of cGMP in neuronally-derived cells and tissues. Our current studies are directed at determining the site of action of cAMP and the localization of the cAMP receptor which mediates G2 arrest